An art of optical microscopes is old-established and a variety of types of the microscopes have been developed. In addition, with progress of peripheral technologies including a laser technology and an electronic imaging technology, an even more advanced microscope system has been developed in recent years.
On such a background, there is suggested a highly functional microscope which enables not only a control of contrasts of obtained images but also a scientific analysis thereof by illuminating a sample with multi-wavelength lights to induce double resonance absorption process (for example, see Japanese Patent Laid-Open No. 8-184552).
Such a microscope allows for an observation of absorption and fluorescence caused by a specific optical transition by selecting a specific molecule with the double resonance absorption. A principle thereof will be described with reference to FIG. 19 to FIG. 22. FIG. 19 shows an electronic structure of a valence electron orbital of a molecule composing a sample. First, electrons on the valence electron orbital of the molecule in a ground state (S0 state: stable state) shown in FIG. 19 are excited by light of a wavelength λ1, in order to transit it to a first excited state (S1 state). Next, the electrons are excited by light of a wavelength λ2 in a similar manner, in order to transit it to a second excited state (S2 state) shown in FIG. 21. In this excited state, the molecule emits fluorescence or phosphorescence and returns to the ground state as shown in FIG. 22.
Microscopy adopting the double resonance absorption process is for observing absorption images and emission images by using an absorption process in FIG. 21 and light emission of such as fluorescence and phosphorescence. According to this microscopy, first, the molecule composing the sample is excited to the S1 state by laser light or the like of a resonant wavelength λ1, as shown in FIG. 20. At this time, the number of molecules in the S1 state in a unit cubic volume increases in proportion to an intensity of the light emitted.
Here, a linear absorption coefficient is obtained by multiplying an absorption cross-section per molecule by the number of molecules in the unit cubic volume. Therefore, in an excitation process as shown in FIG. 21, the linear absorption coefficient to light of the resonant wavelength λ2 irradiated subsequently depends on an intensity of the light of the resonant wavelength λ1 initially irradiated. That is, the linear absorption coefficient λ2 to the wavelength λ2 can be controlled by the intensity of the light of the resonant wavelength λ1. This indicates that, by illuminating the sample with the light of the wavelength λ1 and the light of the wavelength λ2 and taking a transmission image by the wavelength λ2, it is possible to control contrasts of the transmission image completely by the light of the wavelength λ1.
In addition, if it is possible to carry out a deexcitation process from the excited state in FIG. 21 to the ground state in FIG. 22 by using fluorescence or phosphorescence, a emission intensity thereof is proportional to the number of molecules in the S1 state. Accordingly, it is possible to control the contrast of the image in using the microscope as a fluorescence microscope, too.
Moreover, the microscopy adopting the double resonance absorption process can perform not only control of contrasts of images as stated above but also chemical analysis. That is, since an orbital of an outermost electron shown in FIG. 19 has an energy level specific to each molecule, the wavelength λ1 is varied among the molecules and, simultaneously, the wavelength λ2 is also specific to the molecules.
Here, even if the sample is irradiated by conventional light of a single wavelength, it is possible, to some degrees, to observe an absorption image or a fluorescent image of particular molecules. Generally, however, since wavelength ranges of absorption bands of some molecules overlap one another, it is not possible to precisely identify a chemical composition of a sample when the sample is irradiated by the light of the single wavelength.
In contrast, the microscopy adopting the double resonance absorption process limits molecules to absorb or to emit light by using two wavelength, λ1 and λ2, which enables more precise identification of the chemical composition of the sample than conventional methods. In addition, in excitation of the valence electron only the light with a particular electric field vector relative to a molecular axis is intensely absorbed, therefore taking the absorption image or the fluorescence image by deciding a polarization direction of the light of the wavelength λ1 and the light of the wavelength λ2 enables identification of orientation directions of the same molecules.
Additionally, there is recently suggested a fluorescence microscope highly capable of a spatial resolution exceeding a diffraction limit by adopting the double resonance absorption process (for example, see Japanese Patent Laid-Open No. 2001-100102).
FIG. 23 shows a conceptual diagram of the double resonance absorption process of the molecule, in which the molecule in the ground state S0 is excited by the light of the wavelength λ1 to the first excited state and further excited by the light of the wavelength λ2 to the second excited state S2. It is to be noted that FIG. 23 shows that fluorescence from a molecule of a certain type in the S2 state is extremely weak.
The molecule with an optical property as shown in FIG. 23 presents a very interesting phenomenon. Like FIG. 23, FIG. 24 is also a conceptual diagram of the double resonance absorption process which shows a vertical axis X representing an expansion of a spatial distance, a spatial area A1 irradiated by the light of the wavelength λ2, and a spatial area A0 not irradiated by the light of the wavelength λ2.
In FIG. 24, numerous molecules in the S1 state are generated by excitation with the light of the wavelength λ1 in the spatial area A0 and, at that time, fluorescence from the spatial area A0 emitted by the light of a wavelength λ3 may be observed. However, since the light of the wavelength λ2 is irradiated to the spatial area A1, most of the molecules in the first excited state S1 are immediately excited to a higher state, the second excited state S2, leaving no molecules in the first excited state S1. This phenomenon is identified for some molecules. Because of this phenomenon, since fluorescence of the wavelength λ3 is completely eliminated in the spatial area A1 and there is no fluorescence from the second excited state S2 from the beginning, fluorescence itself is completely suppressed (fluorescence suppression effect) in the spatial area A1 and fluorescence only from the spatial area A0 is emitted.
In addition, when the light of the wavelength λ2 overlaps a fluorescence emission band, the molecule is forced to transit from the first excited state S1 to a higher vibration level of the ground state S0 by induced emission process. Thus, the fluorescence suppression effect is more enhanced. In other words, with emission of the light of the wavelength λ2, a fluorescence yield emitted from the first excited state S1 is reduced. Accordingly, the fluorescence suppression effect is presented if the molecule is forced to transit to a quantum level. Materials having such properties are photochromic molecules, fluorescent substances including rare earth, quantum dots and the like.
Such a phenomenon has a very important meaning from a point of view of an application field of the microscope. That is, conventional scanning microscopes and the like condense laser beam into a microbeam by using a collective lens in order to scan on the sample to be observed. At this time, a size of the microbeam falls to a diffraction limit dependent on the numerical aperture of the collective lens and the wavelength. Therefore, further spatial resolution cannot be expected in principle.
In a case shown in FIG. 24, however, since the fluorescence area is controlled by spatially partially overlapped two different light of the wavelength λ1 and the light of the wavelength λ2, it is possible, when focusing attention on the emission area of the light of the wavelength λ1, for example, to have the fluorescence area narrower than the diffraction limit depending on the numerical aperture of the collective lens and the wavelength, which leads to a substantial improvement in the spatial resolution. Accordingly, by taking advantage of such a principle, it is possible to substantialize an ultra-high resolution microscope adopting the double resonant absorption process exceeding the diffraction-limited resolution, that is, an ultra-high resolution fluorescence microscope, for example.
In using rhodamine 6G, for example, if light of a wavelength 532 nm (pumping light; first illumination light) is emitted, rhodamine 6G molecules are excited from the ground state S0 to the first excited state S1 and emit fluorescence with a peak at a wavelength of 560 nm. At this time, if light of a wavelength 599 nm (erasing light; second illumination light) is irradiated it causes the double resonance absorption process, rendering the rhodamine 6G molecules transit to the second excited state, in which fluorescent emission is difficult. More specifically, simultaneous irradiation of the pumping light and erasing light to rhodamine 6G suppresses fluorescence.
FIG. 25 is a main section configuration diagram of the ultra-high resolution microscope conventionally suggested. This ultra-high resolution microscope is based on a usual fluorescence microscope of laser scanning type and comprising three independent units, that is, a light source unit 210, a scanning unit 230 and a microscope unit 250.
The light source unit 210 has a pumping light source 211 and an erasing light source 212. The pumping light emitted from the pumping light source 211 is incident to a dichroic prism 213 and is reflected thereby. The erasing light emitted from the erasing light source 212 is incident to the dichroic prism 213 after being subjected to spatial modulation of its phase by a modulation optical element 215, transmits through the dichroic prism 213 and then exits as combined concentrically with the pumping light.
Here, in observing the sample dyed with rhodamine 6G the pumping light source 211 is configured, using Nd:YAG laser, to emit the light of the wavelength 532 nm, which is second harmonic waves of the laser. In addition, the erasing light source 212 is configured, using Nd:YAG laser and Raman shifter, to emit light, which is second harmonic waves of the Nd:YAG laser modulated into the light of the wavelength 599 nm by the Raman shifter, as the erasing light.
The modulation optical element 215 modulates the phase of the erasing light and has a pupil plane radially divided into 8 regions about an optical axis as shown in FIG. 26, for example. Each of the regions is formed by forming optical multilayer films having phases different by λ/8 of the wavelength of the erasing light from one another such that a phase difference of the erasing light revolves by 2π about the optical axis or by etching a glass substrate. When the erasing light having transmitted through the modulation optical element 215 is collected, it generates a hollow erasing light canceling the electric filed on the optical axis.
The scanning unit 230, after passing the pumping light and the erasing light coaxially emitted from the light source unit 210 through a half prism 231, performs swing scanning in a two-dimensional directions with two galvano mirrors 232, 233 in order to emit the lights to the microscope unit 250, which will be described below. In addition, the scanning unit 230 branches fluorescence incident from the microscope unit 250 tracking back its path by using the half prism 231, such that branched fluorescence is received by a photomultiplier 238 via a projector lens 234, a pinhole 235, and notch filters 236, 237.
For the sake of simplification of the diagram, the galvano mirrors 232, 233 are swingable in a coplanar manner in FIG. 25. The notch filters 236, 237 eliminate the pumping light and the erasing light mixed into fluorescence. In addition, the pinhole 235 is an important optical element composing a confocal optical system and passes only fluorescence emitted on a particular cross-section in the sample being observed.
The microscope unit 250 is a usual fluorescence microscope which reflects the pumping light and the erasing light incident from the scanning unit 230 on the half prism 251 and collects the lights, by using a microscope objective lens 252, on a sample to be observed containing molecules with three electron states including at least the ground state. In addition, fluorescence emitted on a sample 253 is collimated by the objective lens 252 again and reflected on the half prism 251 so as to return to the scanning unit 230, while a part of fluorescence passing the half prism 251 is led to an eyepiece 254 so as to be visually observed as a fluorescence image.
According to this ultra-high resolution microscope, fluorescence except the same close to the optical axis, at which the intensity of the erasing light becomes zero on a focusing point of the sample 253, is controlled and, as a result, it enables to measure only fluorescence labeler molecules in a region narrower than a width of the pumping light. Accordingly, by arranging fluorescent signals at each measurement point in the two-dimension on a computer, it is possible to form a microscopic image having a resolution exceeding the spatial resolution of the diffraction limit.
It is to be noted that the modulation optical element may be configured to modulate polarization of the erasing light in order to generate a hollow erasing light canceling the electric field on the optical axis (for example, see Y. Iketaki, et. al, Rev. Sci. Instrum. 75 (2004)5131). In addition, the modulation optical element may be disposed on a common optical path of the pumping light and the erasing light (for example, see Japanese Patent Laid-Open No. 2010-15026). In this case, the modulation optical element is formed into an annular shape having a center region and a peripheral region separated concentrically. The center region has optical multilayer formed on a transparent optical substrate, such as a glass substrate or the like, to reflect the pumping light while making the erasing light transmit therethrough by inverting the phase by π. The peripheral region is formed of an optical substrate, for example, and makes the pumping light and the erasing light transmit therethrough without phase modulation. Alternatively, the modulation optical element has a plurality of regions radially divided about the optical axis, each of which is formed of the optical multilayer for making the pumping light transmit in the same phase while modulating the phase of the erasing light in order to form a Laguerre-Gaussian beam with a phase distribution revolving by 2π.